Liver Assessment in Patients with Ataxia-Telangiectasia: Transient Elastography Detects Early Stages of Steatosis and Fibrosis

Background Ataxia-telangiectasia (A-T) is a rare autosomal-recessive multisystem disorder characterized by pronounced cerebellar ataxia, telangiectasia, cancer predisposition, and altered body composition. Liver diseases with steatosis, fibrosis, and hepatocellular carcinoma are frequent findings in older patients but sensitive noninvasive diagnostic tools are lacking. Objectives To determine the sensitivity of transient elastography (TE) as a screening tool for early hepatic tissue changes and serum biomarkers for liver disease. Methods Thirty-one A-T patients aged 2 to 25 years were examined prospectively from 2016–2018 by TE. In addition, we evaluated the diagnostic performance of liver biomarkers for steatosis and necroinflammatory activity (SteatoTest and ActiTest, Biopredictive, Paris) compared to TE. For calculation and comparison, patients were divided into two groups (<12, >12 years of age). Results TE revealed steatosis in 2/21 (10%) younger patients compared to 9/10 (90%) older patients. Fibrosis was present in 3/10 (30%) older patients as assessed by TE. We found a significant correlation of steatosis with SteatoTest, alpha-fetoprotein (AFP), HbA1c, and triglycerides. Liver stiffness correlated significantly with SteatoTest, ActiTest, HbA1c, and triglycerides. Conclusion Liver disease is a common finding in older A-T patients. TE is an objective measure to detect early stages of steatosis and fibrosis. SteatoTest and ActiTest are a good diagnostic assessment for steatosis and necroinflammatory activity in patients with A-T and confirmed the TE results.

Hepatopathy in A-T is usually mild and does not lead to a limitation of the synthesis or detoxifcation function of the liver [7]; however, up to 92.9% of older A-T patients are afected [14]. Liver disease certainly belongs to the complex phenotype of premature aging [19], which also includes insulin resistance (IR), diabetes mellitus type 2 [6,20], and dyslipidemia and thus leads to an incomplete metabolic syndrome. All these factors naturally favor fatty remodeling of the liver with a consecutive increase in liver enzymes. In addition, a few severe cases of liver failure and HCC have been published as case reports on A-T patients [15,17].
One of the central aims of modern hepatology is the search for noninvasive diagnostic procedures for presymptomatic liver diseases in at-risk patient groups. In order to prevent severe courses and identify critical patients at an early stage, sensitive, fast, and minimally invasive screening tools are needed to monitor the course of the disease [21,22]. Currently, a liver biopsy is the gold standard for assessing the severity of nonalcoholic fatty liver disease (NAFLD), NASH, and the stage of liver fbrosis. Nevertheless, limitations and complications include invasiveness, severe bleeding, sampling error, and pneumothorax [23].
Transient elastography (TE) is a precise, noninvasive method to determine the extent of fbrosis and the degree of fatty degeneration of the liver [24]. In contrast to biopsy, it is painless and noninvasive. In addition, it records a multiple of the parenchyma. Te classifcation into fbrosis and steatosis stages is objective in comparison to normal sonography, as it is carried out using fxed cutof values [22,24,25]. Measurements are reproducible and independent of the user [25,26].
In this cross-sectional study, we prospectively evaluated liver assessment by TE and liver scores (FibroMax) for A-Tassociated liver disease to predict the extent of liver disease and identify at-risk patients for severe disease courses.

Patients and Methods
Te current study is a prospective, cross-sectional, clinical, single-center trial.

Patients.
From November 2016 to May 2018, 31 patients with a clinically and/or genetically confrmed diagnosis of A-T aged between two and 25 years were included in the study (Table 1). Malignancy and clinical and laboratory-associated infections were defned as the exclusion criteria.
Te Ethics Committee of University Hospital Frankfurt approved the trial (Reference No. 504/15). Te study was registered at clinicaltrials.gov (NCT03357978). One study visit was conducted. Written consent was obtained from all patients and/or caregivers. Te study was conducted according to the ethical principles of the Declaration of Helsinki and regulatory requirements and the code of Good Clinical Practice.

Transient Elastography.
Te examination was performed with FibroScan ® (Echosens, Paris, France). Te examination probe is formed by a vibration generator and an ultrasonic probe (3.5 MHz) aligned on the same axis. Te vibration generator oscillates at a frequency of 50 Hz, which leads to shear waves in the liver tissue. Te speed of propagation of this shear wave correlates directly with liver stifness and therefore with the extent of fbrosis. Te result of this liver stifness measurement (LSM) is given in kilopascals (kPa) [25].
Te interpretation of the measurement results was based on the limit values of a study on mixed hepatopathy by Fraquelli et al. [26]. A distinction was made between three fbrosis stages: F ≥ 2 � pronounced fbrosis, F ≥ 3 � severe fbrosis, and F4 � cirrhosis.
At the same time, the controlled attenuation parameter (CAP) was measured using the same signals. Te attenuation of the ultrasound signal (3.5 MHz) in the liver is measured in dB/m. Te attenuation correlates with the degree of liver steatosis [27]. Te diferent stages of steatosis were defned as follows: S ≥ 1 � steatosis in 11-33% of hepatocytes, S ≥ 2 � steatosis in 34-66% of hepatocytes, and S � 3 steatosis in 67-100% of hepatocytes. Te cutof values proposed by Karlas et al. in 2017 were used [28].
Te examination was performed by an experienced physician in the supine position and maximum abduction of the right arm through a right intercostal space. In order to perform a standardized measurement, patients were asked to fast for at least 4 hours before the examination. A success rate of at least 60% or the interquartile range below 30% of the median measurement result was considered necessary.
Te accuracy of the measurement may be reduced in obesity or ascites.
Te FibroMax data were calculated by BioPredictive (Paris, France) using a patented algorithm.

Statistical Analysis.
For statistical analysis, GraphPad Prism 5.01 (GraphPad Software, Inc.) was used. Values are presented as arithmetic means with standard deviation (SD). For comparisons between the two study groups, the twotailed Mann-Whitney U test was applied. Correlations were analyzed by Spearman's correlation coefcient. p values ≤ 0.05 were considered signifcant.

Results
In a study period of 19 months (November 2016 to May 2018), 31 patients with A-T were examined. Patients' characteristics are shown in Table 1. Te age distribution ranged from 2 to 25 years (mean age: 10.7 years). Te patients were divided into the two groups for evaluation. Twenty-one patients were <12 years (group 1), and ten patients were ≥12 years (group 2). No patient had a history of infectious hepatitis or was taking hepatotoxic drugs regularly.

Serum Biomarkers and Liver Scores.
A complete data set for biomarkers was available for 30 patients. Te results of FibroMax are shown in Table 2. Signifcantly lower values for group 1 concerning SteatoTest and ActiTest (SteatoTest: p < 0.0001; ActiTest: p < 0.001) were calculated, showing a normal function in group 1 and mild to moderate dysfunction in group 2. FibroTest did not show signifcant diferences between the two groups, with normal to slightly elevated levels in all patients.
Te number of patients in each category of SteatoTest, ActiTest, and FibroTest is shown in Supplementary Table 2. Table 4 shows lipid parameters and HbA1c. Four of ten older patients had type 2 diabetes. No diference was found between the two groups in total cholesterol. However, when broken down into HDL and LDL cholesterol, the older group showed signifcantly lower HDL cholesterol and signifcantly higher LDL cholesterol values (HDL cholesterol: p ≤ 0.001; LDL cholesterol: p ≤ 0.05).

Examination of Metabolic Biomarkers, Infammation, and AFP.
Te LDL cholesterol values of the two groups were within the normal range. HDL cholesterol was lower in 8/20 (40%) younger patients and in all older patients (100%).
Tere was a signifcant diference in the LDL-HDL ratio between the two patient groups (p ≤ 0.0001). Triglycerides were signifcantly increased in group 2 (p < 0.0001). Five of ten (50%) older patients had values above the normal range. In group 1, the triglyceride values were all within the normal range. In addition, there was a signifcant correlation of triglycerides with age (r � 0.66, p ≤ 0.0001).
As expected, AFP was elevated in all patients. However, there was a signifcant correlation of AFP values with age (r � 0.54, p < 0.01).
For CRP, no signifcant diference between the two groups was found.

Discussion
A-T is a life-limiting systemic disease clinically characterized by neurodegeneration, radiosensitivity, increased risk of malignancy, immunodefciency, failure to thrive, and hepatopathy [1, 3, 9-11, 14, 33]. To date, the clinical signifcance of liver disease is unclear, but up to over 90% of patients develop an elevation of liver enzymes with advancing age, which is associated with a high degree of fatty degeneration and sometimes fbrosis of the liver tissue [14].
To the best of our knowledge, the present study is the frst prospective trial addressing noninvasive procedures to characterize liver disease in A-Tand relate outcomes of TE to     liver biomarkers and liver scores. Our results demonstrate for the frst time that 10% of younger patients as opposed to 90% of older patients have liver steatosis. In line with this fnding, higher degrees of steatosis were present in the older group. Pronounced fbrosis was found in 3/10 (30%) older patients. In two patients, cirrhosis was already present according to TE. We found a signifcantly higher necroinfammatory activity (ActiTest) in the older patient group. Necroinfammation can be defned as the immune response to necrosis [34]. Terefore, ActiTest is a good marker for the progression of liver disease to steatosis and apoptosis with increased necroinfammatory activity. Te presence of necroinfammatory activity, which also correlates with the CRP value, indicates NASH, which according to our data mainly afects older patients. In summary, SteatoTest and ActiTest are a suitable diagnostic assessment for steatosis and necroinfammatory activity in patients with A-T and have confrmed the TE results. FibroTest did not show a signifcant diference between the two patient groups or a signifcant correlation with age or the results of TE, most likely since FibroMax is not licensed below the age of 14 years due to the physiological increase of some of the serum markers used for calculation.
We were also able to show a signifcant correlation of AFP with steatosis (CAP). However, the mechanism has not been elucidated so far. AFP is mainly known as a tumor marker for HCC. Serum AFP may also be elevated by germ cell tumors, viral hepatitis, liver fbrosis, and neurodegenerative diseases such as A-T [35][36][37]. Among other mechanisms, tumor suppressor p53 acts as a repressor on the AFP gene during development and regeneration of the liver [38][39][40]. Via the reduced activation of p53 due to the absence of the ataxia-telangiectasia-mutated (ATM) kinase, an increased expression of AFP could thus occur in A-T [41]. A mutation of p53 is also frequently found in HCC [42], which could be a possible explanation for an increase in AFP. In contrast to CAP, however, no signifcant correlation of LSM with AFP was found. Te missing correlation of AFP with the LSM could thus be explained by a loss of functional liver tissue in cirrhosis.
Dyslipidemia is common in older A-T patients [6,8]. While triglycerides and LDL cholesterol were signifcantly higher, HDL cholesterol was signifcantly lower in the older patient group. In addition, there was a signifcant correlation  Canadian Journal of Gastroenterology and Hepatology of triglycerides with age. Te association between elevated liver enzymes and dyslipidemia has been described before [6]. We could also show a signifcant correlation of HbA1c and triglycerides with CAP and LSM, emphasizing the effects of metabolic risk factors for liver tissue remodeling. Diabetic metabolism leads to fatty liver remodeling due to hyperglycemia and hypertriglyceridemia and thus increases the risk of NASH. In addition, the described alterations in cholesterol and triglyceride levels in patients indicate an increased atherosclerotic risk profle [43]. Tey could also play an important role in the development of steatosis, through the accumulation of fat in the liver [44]. Te increased infux of free fatty acids to the liver leads to an increased synthesis of triglycerides and very low-density lipoprotein (VLDL), as well as a reduced synthesis of HDL cholesterol [45]. ATM is induced by the accumulation of fat and thus elevates oxidative stress in the liver [46] and acts as an activator of p53, which in turn activates the p53 upregulated modulator of apoptosis (PUMA) [47]. PUMA is a crucial player in steatosis and apoptosis in hepatocytes [48]. Since hepatocyte apoptosis correlates with the severity of NASH and the stage of fbrosis [49], it can be assumed that steatosis related apoptosis is partly responsible for the progression of liver disease [46]. In the absence of ATM, alternative signaling pathways must be activated, which ultimately cause fbrotic remodeling and death of functional liver tissue.
Fibrosis results from connective tissue remodeling due to chronic infammation of the liver tissue [50,51], e.g., in response to steatosis and apoptosis [46]. Te progression of fbrosis to cirrhosis of the liver then appears to develop through repetitive phases of infammation and the subsequent reparative immune response [52]. Tis leads to a loss of functional liver tissue, which is replaced by scar tissue [50,53].
ATM can be activated directly by oxidative stress, even independently by double-strand breaks in DNA [54]. Te absence of ATM leads to low antioxidant capacity [55]. As a result, macromolecules, lipids, and DNA are exposed to permanent oxidative stress and damage it causes. Similar results have been shown for NAFLD and in particular NASH, where oxidative stress and lipid peroxidation are also elevated [56]. For example, hepatocytes with excess fat are particularly susceptible to oxidative stress and DNA damage [57]. Tis may be related to the fact that saturated free fatty acids promote the formation of ROS, thereby inducing apoptosis and infammation [58]. It is also known that oxidative stress increases with the severity of liver disease [57]. Oxidative stress and resulting infammation appear to play a major role in the development and progression of liver disease. A link between hepatopathy in A-T and the already known increased oxidative stress levels in patients therefore seems very likely.
Te type of liver involvement in A-T is not yet defned, as the published cases with severe portal hypertension causing ascites and varices had no evidence of cirrhosis at a liver biopsy [15,16,18]. It seems that whether these complications are to be attributed to steatosis/NASH leading to portal hypertension in the absence of cirrhosis [59] or to vascular liver disease/porto-sinusoidal vascular disorder (PSVD) is still open for discussion [60]. In this view, clinicians have to consider the value of a liver biopsy, especially in a research context where little is known about liver disease in A-T (i.e., the diagnosis of PSVD can be histological). Te rate of adverse events in the modern era is negligible, and the information one can obtain is precious.
Te study has some limitations. Due to the size of the study population and the monocentric study design, no general statements can be made. Tere are no corresponding ultrasound reports that can be compared with the TE results. Tis is a major limit, not only because it could have shown how liver disease (i.e., fbrosis staging) would have been underestimated by ultrasound only, but also because signs of portal hypertension (i.e., splenomegaly, dilated portal vein, and collaterals) could have been detected and correlated with the absence/presence of liver cirrhosis at TE. In addition, the application of spleen stifness would have been useful to address more specifcally the severity of portal hypertension, as is complementary to LSM [61], and the spleen stifness measurement (SSM)/LSM ratio could be informative in these cases [62].
Additionally, the examination methods used in this study are not yet established practice in pediatrics, so there are no universally accepted cutof values for children, but several studies have highlighted the value of TE with CAP [63][64][65][66]. Te proposed cutof values are largely consistent with those for adults, which were used in this study. Also FibroTest could show its diagnostic value in some pediatric studies and could distinguish between severe fbrosis and absence of fbrosis [67]. To the best of our knowledge, there are no pediatric studies on FibroMax to date.
Due to the mildness of liver disease, validation of TE by biopsy (gold standard) was not performed. Although CAP is not recommended as a standard tool for stratifcation of hepatic steatosis [68], there are data showing a good correlation with other steatosis markers, and our data also support this. Another limitation is the lack of a healthy control group to compare the results. As only one TE measurement was performed in each patient, no interobserver comparison is possible. For detection of steatosis by TE, larger cohorts and multicenter studies are needed in order to validate the clinical application.

Conclusion
Premature aging, low-grade infammation, oxidative stress, dyslipidemia, and IR as common features of A-T contribute to the development of NAFLD. Liver disease in the context of A-T should be monitored regularly in order to prevent long-term consequences, such as NASH, cirrhosis, and HCC. TE and liver scores are a well reproducible, noninvasive method detecting early stages of liver disease. SteatoTest and ActiTest are useful to assess steatosis and necroinfammatory activity in patients with A-T and have confrmed the TE results. 6 Canadian Journal of Gastroenterology and Hepatology